The field of the invention is oxymetry, and particularly, the measurement of oxygen concentration using electron spin resonance (ESR) spectrometers.
ESR spectrometers measure electron paramagnetic resonance signals. The Larmor theorem states that when electrons are subjected to a strong magnetic field they will precess about the axis of the field at a frequency which is directly proportional to the magnetic field strength. If a sample to be analyzed is subjected to a strong d.c. "polarizing" magnetic field and is at the same time irradiated by a radio frequency "source" magnetic field at the electron's frequency of precession, then electron resonance occurs. ESR spectrometers observe the effects of this resonance.
ESR spectrometers produce electron resonance by applying a radio frequency source magnetic field to the sample material while it is disposed within a polarizing magnetic field. A low frequency "reference" magnetic field modulates the polarizing magnetic field causing the sample material to pass through gyromagnetic resonance during each cycle of the reference field. At resonance the sample material produces a radio frequency signal which has components that are in-phase with the applied radio frequency source field and components that are ninety degrees out of phase with the source field. These components may be detected separately by ESR spectrometers when the spectrometer is operated in the "absorption mode" or the "dispersion mode." These two signal modes are referred to as the "v" and "u" modes in a notation introduced by F. Bloch, in the Physical Review, Volume 70, page 460, 1946. These signal modes are also known as the imaginary and real parts of the complex magnetic susceptibility. When gyromagnetic resonance occurs, the absorption signal mode is accompanied by a change in the resonator Q, or quality factor, and the dispersion signal mode is accompanied by a change in the resonant frequency of the resonator. At a detection point, as for example, a microwave diode, where the radio frequency source signal is rectified, the absorption and dispersion signal modes are characterized as radio frequency voltages in quadrature with each other. These two phase-displaced signals can be detected separately by applying a radio frequency signal to the detector which is "in-phase" with the source signal or by applying a radio frequency signal to the detector which "out-of-phase", or in quadrature, with the source signal.
The absorption mode signals and the dispersion mode signals both provide specific information concerning the structure and changes of the atomic and molecular particles in the sample material. However, an experimental problem has long existed in the detection of the dispersion mode signal by ESR spectrometers. More specifically, when the spectrometer is set to the dispersion mode, it demodulates the phase noise in the radio frequency source signal. The amount of this demodulated phase noise depends on the Q of the resonator which contains the sample material, the phase noise in the radio frequency source oscillator, and the amount of radio frequency power applied to the sample. This demodulated phase noise of the source signal obscures the gyromagnetic dispersion mode signal. Although a number of solutions have been found to this problem, these solutions are expensive and complex and are only practical in a laboratory environment.
The measurement of dissolved oxygen concentration levels in biological systems and the measurement of the rate of change of oxygen concentration in such systems provides valuable information. Such measurements have been made for a number of years using laboratory ESR spectrometers as reported in The Proceedings of the National Academy of Science USA, Volume 79, pages 1166-1170, published in February 1982. The physical basis for such measurement methods relies on the "Heisenberg" exchange between molecular oxygen in the system and a nitroxide radical spin-label material which is added to the biological system for measurement purposes. One such label is 3-carbamoyl-2, 2, 5, 5-tetramethyl-3-pyrroline-1-yloxyl, known in the art as "CTPO". It is commercially available from Aldrich Chemical Co. This exchange occurs at the bimolecular collision rate (.omega.) determined by the Smoluchowski equation: EQU .omega.=4.pi.RD[O.sub.2 ]
where R is the interaction distance; D is the diffusion constant of molecular oxygen; and [O.sub.2 ] is the concentration of molecular oxygen. The measurement of this exchange rate (.omega.) provides a direct measurement of the diffusion-oxygen concentration product (R is generally assumed to be a constant 4.5 angstroms). In addition, the rate of change of the exchange rate (.omega.) provides a direct measurement of the rate of change of oxygen concentration, since the diffusion constant D remains relatively constant in any given system.
ESR spectrometers have been employed to measure the collision rate (.omega.) between molecular oxygen [O.sub.2 ] and a nitroxide radical spin-label material using a number of methods. In one method the absorption signal produced by small nitroxide radicals freely tumbling in solution is observed. The resolution of this signal, however, tends to disappear in the presence of molecular oxygen at concentrations of biological relevance. This method, referred to in the art as the T.sub.2 oximetric method, thus has a limited range and does not work in viscous environments where the nitroxide radicals are not freely tumbling.
Another ESR spectrometer method for measuring the collision rate (.omega.) between molecular oxygen and nitroxide radical spin-labels may employ measurement of either the absorption or the dispersion signal. This method measures changes in the spin-lattice relaxation time T.sub.1 which is the time it takes for the electrons to return to their unexcited state after the signals which produce electron spin resonance are removed. The T.sub.1 time of the spin-label is mediated by the Heisenberg exchange with molecular oxygen and its measurement provides a sensitive measurement of the collision rate over a broad range of oxygen concentration levels. Although this measurement technique has been employed in the laboratory, the equipment required is too complex and expensive for commercial applications.